Transcription is the first step of gene expression and the step at which the majority of gene expression regulation occurs. The long-term goal of the applicant's research is to understand the steps leading to transcription initiation at a molecular level. Such an understanding is of great importance, not only for advancing our basic knowledge of this fundamental biological process, but also for practical applications; e.g., the rational design of drugs targeted to transcription initiation complexes or the design of better expression systems. This proposal is aimed at filling some important voids in knowledge of steps leading to transcription initiation and is focused on the sigma70 subunit of E. coli RNA polymerase. The sigma70 subunit is essential for initial promoter recognition, is also involved in melting of promoter DNA, and is likely to be an important determinant of productive full-length transcript synthesis. The immediate goals of this proposal are to (1) determine the mechanism of initial recognition of promoter DNA by RNA polymerase holoenzyme, (2) determine the nature of a rate-limiting conformational change in promoter-polymerase complex, and (3) determine the role of sigma70 subunit in the formation of a transcribing complex in the elongation mode. The applicant will use a combination of molecular biology, protein chemistry and fluorescence spectroscopy methods to engineer the components of transcription initiation complexes, to allow selective observation and study of individual steps of transcription initiation. The data are expected to provide the first three-dimensional description of time-dependent structural changes in transcription initiation complex, as it evolves from the initial promoter recognition complex to an elongation complex capable of productive full-length transcript synthesis. The applicant hopes to identify critical determinants of initial promoter recognition and productive elongation, complex formation.